The goal of this project is to gain insight into alterations of cell cycle control mechanisms in neoplastically transformed mammalian cells. A requisite step in the process of carcinogenesis is the loss of normal control of cellular proliferation. This can result, at least in part, from gain-of-function mutations (such as activations of oncogenes), loss-of- function mutations (such as inactivations of normal tumor suppressor gene functions), and both heritable and non-heritable alterations in the normal pattern of gene expression and function. We are studying alterations in growth control signal transduction pathways in genetically altered mammalian cells in culture as compared with their normal counterparts. Specifically, we are investigating cell cycle related parameters in mouse fibroblasts that constitutively overexpress specific oncogene products and in primary embryo cultures from mice with one or both alleles of the p53 tumor suppressor gene inactivated. We have been carefully defining the cell division cycle kinetics of clonal populations of NIH3T3 cells or NIH3T3 cells overexpressing mosmu, mosXe, H-rasva112, tpr-met, v-src, and c-srcmu. These studies involve optimizing synchrony protocols through the use of various combinations of either serum deprivation, mitotic shake-off, hydroxyurea treatment, or aphidicolin treatment. The degree of synchrony is being monitored by such parameters as incorporation of 3H-thymidine or BrdU into newly synthesized DNA, mitotic indices, and population doublings. We are investigating cell cycle related parameters such as the level of expression and phosphorylation status of the cdc2-family of kinases, cyclin proteins, tubulin proteins, reorganization of interphase microtubules to mitotic spindles, and phosphorylation events associated with the activation of microtubule organizing centers (MTOCs) and maturation promoting factor (MPF) activity. Additional studies will follow specific gene products (such as p34cdc2, cyclin proteins, and p53) in genetically altered cells as compared to their normal counterparts as they progress through their division cycle following exposure to environmental genotoxic and non- genotoxic carcinogens.